The present invention relates to a newly discovered and purified protein complex and to methods and compositions including such protein complex in assays and for diagnosing, preventing and/or treating cellular debilitation, derangement or dysfunction. More particularly, the present invention relates to a molecule or molecules that specifically promote S phase activity, and to antibodies or to other entities specific thereto that may thereby selectively inhibit S phase activity of mammalian cells.
The study of DNA replication in a variety of prokaryotic chromosomes, bacteriophages, and eukaryotic viruses has facilitated the construction of a general model for initiation of DNA synthesis at origins of replication (Bramhill, D., & Kornberg, A. (1988). CELL, 54, 915-918). In the general model, initiation is viewed a stepwise process that involves the ordered interaction of multiple proteins with origin DNA sequences. Origin activation commences with the binding of a replication initiator protein to origin-specific recognition sequences, which are often reiterated. Binding of the initiator protein engenders DNA melting at the origin, leading to the formation of a stable presynthesis complex that contains locally unwound DNA (Bramhill & Kornberg, 1988; Dean, F. B. et al. (1987). PROC. NATL. ACAD. SCI. USA, 84, 16-20; Schnos, M. et al. (1988). CELL, 52, 385-395). The presynthesis complex, in turn, fosters the assembly of the multi-enzyme complexes required for bidirectional DNA replication. The timing and frequency of initiation may be regulated by the availability of the initiator protein, or by topological perturbations in the DNA template that affect the ability of the initiator protein to unwind origin sequences (Baker, T. A., & Kornberg, A. 1988). CELL, 55, 113-123). Other mechanisms of initiation of DNA synthesis may be extant in eukaryotic cells.
Like transcriptional promoters, origins of replication are complex regulatory elements with multiple modular components, including DNA unwinding elements (Umek, R. M. et al. (1989). BIOCHIMICA ET BIOPHYSICA ACTA, 1007, 1-14), binding sites for the initiator and accessory factors, and often transcriptional enhancers and promoters (DePamphillis, M. L. (1988). CELL, 52, 635-638). Despite the diversity of organization among origins of replication, unwinding at the origin is likely to be a universal prerequisite for initiation of bidirectional DNA synthesis on duplex DNA templates.
Both DNA sequences and proteins contribute to DNA unwinding at origins of replication. The DNA sequences that facilitate origin unwinding, including DNA unwinding elements, are generally AT-rich, and may have special structural properties. Another structural element, stably bent DNA, is a functional component of several known replication origins, in bacteriophages, yeasts, and papovaviruses. DNA bending may promote helix disruption, foster the functional interaction of protein binding sites, or serve to uniquely orient the topology of origins for subsequent activation of events. The relationship between those origin sequences that promote DNA bending and those that permit facile DNA unwinding is as yet unknown.
In addition to sequence determinants that promote helix instability, extensive unwinding of origin DNA requires the action of a DNA helicase. The helicase activity may be an inherent property of the initiator protein as in SV40 large T-antigen (Stahl, H. et al. (1986). EMBO. J., 5, 1939-1944) and the Herpes Simplex Virus UL-9 protein (Olivo, P. D. et al. (1988). PROC. NATL. ACAD. SCI. USA, 85, 5414-5418), or may comprise a distinct factor that interacts in a specific fashion with the initiator protein-origin DNA complex. The helicase involved in initiation may also be required at replication forks during the elongation phase of DNA synthesis, and therefore might not represent an initiation-specific replication factor.
Insight into the mechanisms that regulate initiation of DNA synthesis in higher eukaryotic cells requires knowledge of the DNA sequences that comprise chromosomal origins of replication, and purification of the cognate factors that interact with origin sequences during entry into the S phase. Although extensive genetic, biochemical and physical evidence suggests that DNA synthesis begins at preferred sites in higher eukaryotes, the lack of a satisfactory, functional assay has greatly impeded the isolation of origins of replication from animal cells, and accordingly no one has yet identified the proteins or enzymes which initiate replication of chromosomal DNA in higher eukaryotes or animal cells.
A need therefore exists to further elucidate the structure and activity of the replication initiator for animal cells, and to derive therefrom improved diagnostic techniques, and therapeutic modalities that offer the promise of greater efficacy and concomitantly reduced risk of injury to non-target cells.